Throughout this application, various publications are cited by Arabic numerals. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the state of the art to which this invention pertains.
Uterine leiomyomas (also known as fibroids) are the most common pelvic non-malignant tumors in women, with incidence ranging from 20%-30% in clinical diagnoses of women of reproductive age to 50% in studies conducted at autopsy (1). Uterine fibroids cause abnormal uterine bleeding and pelvic pressure or pain. Presently, hysterectomy is the only cure for leiomyomas, although recent studies have shown that hormonal treatments such as gonadotropin-releasing hormone agonists or the antiprogesterone RU 486 can reduce uterine leiomyoma size by 34%-61% (2-5). Despite the major importance of leiomyomas in clinical gynecology, little is known about their basic biology and the regulation of their growth.
Uterine leiomyomas arise from a single cell, and the resulting tumors grow rapidly and become fibrotic following excess extracellular matrix formation. Analysis of individual fibroids have shown that cells within the tumor exhibit one variant of glucose-6-phosphate dehydrogenase, phosphoglycerokinase, and uniform restriction fragment length polymorphisms of X-chromosomes, verifying that these tumors have a unicellular origin (6-8). Clinical reports have shown that leiomyomas can grow rapidly, particularly during progestin, clomiphene, or tamoxifen therapy (9, 10). The fibrotic nature of the tumor develops as a result of extensive extracellular matrix deposits, which contain greater levels of collagen and proteoglycan than does the myometrium (6, 11). The presence of increased deposits of extracellular matrix in uterine leiomyomas suggests that regulatory mechanisms involved in matrix formation and remodeling may be inappropriately controlled.
Matrix metalloproteinases are enzymes that have been shown to be present in both the epithelial and stromal layers of the normal endometrium during phases of the menstrual cycle when remodeling occurs. The levels of mRNA for several metalloproteinases, including stromelysin-1, stromelysin-2, stromelysin-3 and matrilysin, have been shown to be elevated in the endometrium during the late secretory-menstrual phase and during the early proliferative phase of the cycle (12-14). It is generally accepted that expression of metalloproteinases in the endometrium is coordinated by and dependent upon gonadal steroids.
Similar to the gonadal steroid-dependent growth of the endometrium, myometrial growth is influenced by changes in estrogen and progesterone levels. Removing gonadal steroids by treating patients with GnRH agonists so as to induce a hypoestrogenic state resulted in a reduction of the size of the fibroid and in the entire uterus, while discontinuing GnRH therapy resulted in regrowth of the leiomyoma and the uterus (2, 3). Progesterone has also been shown to promote growth of uterine fibroids (15), and treatment with the antiprogesterone RU 486 has been shown to decrease fibroid size by 49% (5, 16). While the involvement of metalloproteinases in normal or pathologic myometrium is unknown, metalloproteinases are implicated in the steroid-dependent growth of leiomyomas and in the aberrant extracellular matrix formation in leiomyomas compared to myometrium.
Endogenous tissue inhibitors of metalloproteinases (TIMP""s) have been shown in many studies to inhibit the catalytic activity of the metalloproteinases. Increases in metalloproteinase expression are often paralleled by low levels of TIMP expression in several diseases including arthritis (17, 18) and invasive cancers (19, 20, 21). The levels of TIMP-1 and TIMP-2 mRNA in the endometrium of women are elevated in the stroma and epithelium of the endometrium during the late secretory and menstrual phases of the menstrual cycle (13, 22). Although metalloproteinases have also been reported to be elevated during these stages of the menstrual cycle, the ratio of TIMP to metalloproteinase may be critical in determining the extent of tissue remodeling in a given tissue. The levels of TIMP-1 and TIMP-2 in leiomyoma and unaffected myometrium are presently unknown.
Therefore, from the art, one of ordinary skill might infer the possibility that metalloproteinases are involved in leiomyoma formation, as evidenced by the steroid-dependent growth of leiomyomas, and further by the lack of normal regulation of extracellular matrix formation in leiomyomas compared to myometrium.
However, despite this possibility and the role of metalloproteinases in endometrial remodeling, one of ordinary skill could not reasonably expect that metalloproteinases in general are involved in leiomyoma formation. Moreover, one of ordinary skill could not reasonably expect that certain metalloproteinases, and not others, are involved in leiomyoma formation.
This invention provides a method of treating a subject suffering from a leiomyoma which comprises administering to the subject a therapeutically effective dose of an agent which specifically inhibits at least one metalloproteinase selected from the group consisting of stromelysin-2, stromelysin-3 and matrilysin.
This invention also provides a pharmaceutical composition for treating a subject suffering from a leiomyoma which comprises an agent which specifically inhibits at least one metalloproteinase selected from the group consisting of stromelysin-2, stromelysin-3 and matrilysin, and a pharmaceutically acceptable carrier.
This invention further provides a method of treating a subject suffering from a leiomyoma which comprises administering to the subject a therapeutically effective dose of the instant pharmaceutical composition.
This invention further provides a method of treating a subject suffering from a leiomyoma which comprises administering to the subject a therapeutically effective dose of a plurality of agents, each of which specifically inhibits at least one metalloproteinase selected from the group consisting of stromelysin-2, stromelysin-3 and matrilysin.
This invention further provides a pharmaceutical composition for treating a subject suffering from a leiomyoma which comprises a plurality of agents, each of which specifically inhibits at least one metalloproteinase selected from the group consisting of stromelysin-2, stromelysin-3 and matrilysin, and a pharmaceutically acceptable carrier.
This invention further provides a method of treating a subject suffering from a leiomyoma which comprises administering to the subject a therapeutically effective dose of the instant pharmaceutical composition.
This invention further provides a method of determining whether a tumor in a subject is a leiomyoma, which comprises the steps of (a) obtaining a sample of the tumor from the subject; (b) determining the amount of at least one metalloproteinase present in the sample, the metalloproteinase being selected from the group consisting of stromelysin-2, stromelysin-3 and matrilysin; and (c) comparing the amount of each metalloproteinase determined in step (b) to a known standard, thereby determining whether the tumor is a leiomyoma.
This invention further provides a method of determining whether a tumor in a subject is a leiomyoma, which comprises the steps of (a) obtaining a sample of the tumor from the subject; (b) determining the amount of at least one metalloproteinase-encoding mRNA present in the sample, the metalloproteinase being selected from the group consisting of stromelysin-2, stromelysin-3 and matrilysin; and (c) comparing the amount of each metalloproteinase-encoding mRNA determined in step (b) to a known standard, thereby determining whether the tumor is a leiomyoma.
This invention further provides a kit for use in determining whether a tumor in a subject is a leiomyoma, which comprises (a) an agent suitable for determining the amount of a metalloproteinase present in a tumor sample taken from the subject, the metalloproteinase being selected from the group consisting of stromelysin-2, stromelysin-3 and matrilysin; and (b) a known standard with which the amount of each metalloproteinase determined in step (a) can be compared, so as to permit determining whether the tumor is a leiomyoma
This invention further provides a kit for use in determining whether a tumor in a subject is a leiomyoma, which comprises (a) an agent suitable for determining the amount of a metalloproteinase-encoding mRNA present in a tumor sample taken from the subject, the metalloproteinase being selected from the group consisting of stromelysin-2, stromelysin-3 and matrilysin; and (b) a known standard with which the amount of each metalloproteinase-encoding mRNA determined in step (a) can be compared, so as to permit determining whether the tumor is a leiomyoma.
Finally, this invention provides a method of determining whether an agent specifically inhibits at least one metalloproteinase selected from the group consisting of stromelysin-2, stromelysin-3 and matrilysin, which comprises determining whether the agent (a) slows the reaction rate of stromelysin-2, stromelysin-3 or matrilysin by a factor of at least 50, and (b) does not slow the reaction rate of any metalloproteinase, other than stromelysin-2, stromelysin-3, matrilysin, and gelatinase a and b, by more than a factor of 25, whereby an agent satisfying the criteria of parts (a) and (b) is one which specifically inhibits at least one metalloproteinase selected from the group consisting of stromelysin-2, stromelysin-3 and matrilysin.